Dissertation
Dissertation > Medicine, health > Neurology and psychiatry > Psychiatry > Mental disorders of toxic > Drug-induced mental disorders

Effect of [Glu3, 4, 7, 10, 14]-conantokin G on the Expression of Morphine Induced Conditioned Place Preference in Mice: Role of ERK1/2

Author RenZhanHong
Tutor ZhuYongPing
School Zhejiang University
Course Health Toxicology
Keywords Conditioned place preference(CPP) Morphine Drug dependence Conotoxin [Glu3,4,7,10,14]-conantokin G extracellular signal-regulated kinase1/2(ERK1/2)
CLC R749.61
Type Master's thesis
Year 2008
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1 ObjectiveOne of a most serious issue in today’s society is opioid addiction, which is a chronic and relapsing neurological disease. Although present methods could lessen the withdrawal symptoms, but the replasing rate is still high due to the psychological dependence of opioid drugs.Researches demonstrated that NMDA receptors are involved in opioid dependence, competitive and non-competitive NMDA receptors antagonists could inhibit the conditioned place preference induced by morphine, cocaine and amphetamine.In the present study, we investigated the effect of [Glu3,4,7,10,14]-conantokinG on the expression of CPP induced by morphine in mice, using the computer based video tracking CPP system. We use [Glu3,4,7,10,14]-conantokin G as the entry point to testify NMDA receptor involved in morphine-induced CPP, and further discuss morphine psychological dependence based on these results. Furthermore, the role of ERK1/2 which plays in relative encephalic regions of morphine addiction will be approached.2 Materials and methods2.1 Animals:Adult male Kun-Ming mice (18-22 g) were supplied by Shanghai Centre of Experimental Animals, Chinese Academy of Sciences.2.2 Chemical reagents:Morphine hydrochloride (First Pharmaceutical Company of ShenYang, China); [Glu3,4,7,10,14]-conantokin G (Sigma) ;Anti-p-ERK1/2 polyclonal antibody (Cell Signal);Anti-ERK1/2 polyclonal antibody (Cell Signal);Monoclonal Anti-β-Actin(Sigma);Goat anti-rabbit-IgG/HRP (Biosynthesis biotechnology Co., LTD.).2.3 Apparatus:computer based video tracking CPP system BIO-RAD electrophoresis apparatus Rat/MiceTracing software Quantity-One software SPSS software2.4 Methods2.4.1 Effect of Glu-ConG on the expression of CPP induced by morphine: Male Kun-Ming mice were administered morphine (5 mg/kg, i.p.) in white chamber for 50 min on day1,3,5,7; On day2,4,6,8 mice were given saline in black chamber for 50 min. The control group was treated with a daily saline injection for eight consecutive days both in the morphine and saline paired chambers. To test the effects of [Glu2,4,7,10,14]-conantokinG on the expression of morphine-induced CPP, different dose of [Glu3,4,7,10,14]-conantokinG (0.30、60、120pmol) were given by intracranial administration 30 min before the test on day 9. Total volume was 2μl.2.4.2 Western blotting detection for p-ERK1/2: Hippocampus (HP), prefrontal cortex (PFC) and striatum were separated for total proteins concentration detection.Protocols of western blotting1. Preparation of Polyacrylamide resolving gel and stacking gel;2. Adding sample buffers and flash spinning the samples;3. Samples with equal amounts of protein and the prestained marker were loaded into the wells;4. Run the gel with constant voltage, 70 V for 1.5 h;5. Proteins were transferred to NC membrane, 300mA for 2 h;6. Membranes were incubated in Tris-buffered saline with 0.5% Tween 20 containing 5% non-fat milk for 3 hours at room temperature;7. Incubated with polyclonal rabbit anti-p-ERK1/2 (1:2000) antibodies at 4℃overnight; 8. Wash the membrane 30 min in TBST buffer;9. Incubated with the secondary antibody, goat anti-rabbit IgG conjugated with horseradish peroxidase at a 1:5000 dilution in blocking buffer for 2 h;10.The membranes were then washed 30 min in TBST buffer;11.The blots were developed by enhanced chemiluminescence method;12.The membranes were then washed 30 min in stripping buffer at 50℃13.The membranes were incubated in Tris-buffered saline with 0.5% Tween 20 containing 5% non-fat milk for 3 hours at room temperature;14.Incubated with polyclonal rabbit anti-ERK1/2 (1:2000) antibodies at 4℃overnight;15.Wash the membrane 30 min in TBST buffer;16.Incubated with the secondary antibody, goat anti-rabbit IgG conjugated with horseradish peroxidase at a 1:5000 dilution in blocking buffer for 2 h;17.The membranes were then washed 30 min in TBST buffer;18.The blots were developed by enhanced chemiluminescence method;19.The membranes were then washed 30 min in TBST buffer;20.Incubated with monoclonal rabbit anti-Actin (1:5000) antibodies for 2 h;21.Wash the membrane 30 min in TBST buffer;22.Incubated with the secondary antibody, goat anti-mouse IgG conjugated with horseradish peroxidase at a 1:5000 dilution in blocking buffer for 2 h;23.The membranes were then washed 30 min in TBST buffer;24.The blots were developed by enhanced chemiluminescence method;25.Visualized by exposure to GS-800 Calibrated Densitometer (Bio-Rad).2.5 Data analysis and statisticsAll the data was expressed as the mean±SD. Place Preference index includes the time spent in the drug-paired side; Locomotor Activity index includes the distance that mice moved in chamber or different area; Exploring Activity index includes shuttle times; the total distance that mice moved in the center area of both chambers; the total time spent in the center area of both chambers; the distance that mice moved in the center area of white chamber; the time spent in the cener area of white chamber. A value of P<0.05 was considered significant. The densitometry*mm2 of the bands was calculated by the Biorad quantity one densitomonter, P<0.05 were considered significant.3.Results: 3.1 The acquisition of morphine induced conditioned place preference:Consecutive 3 days preconditioning tests showed that: mice preferred black chamber than white chamber(in the first day, the time spent in balck chamber and white chamber are 489.90±146.97s and 410.10±146.97s, P<0.05; in the second day, the time spent in balck chamber and white chamber are 520.68±120.87s and 379.32±120.87s, P<0.05; in the third day, the time spent in balck chamber and white chamber are 520.50±128.30s and 379.50±128.30s, P<0.05). After 8 days consecutive conditioning trainning, mice in 5mg/kg morphine treatment group preferred white chamber than black chamber(the time spent in balck chamber and white chamber are 313.83±181.20s and 606.82±127.28s, P<0.05).3.2 Effect of [Glu3,4,7,10,14]-conantokin G on the expression of morphine induced CPP:On the CPP expression day(day 9), pretreatment with [Glu3,4,7,10,14]-conantokin G (0, 30, 60 and 120 pmol)dose relatedly suppressed the time spent in white chamber(time spent in white chamber in 0 pmol [Glu3,4,7,10,14]-conantokin G treatment group was 565.4±273.3s, P=0.916; time spent in white chamber in 30 pmol [Glu3,4,7,10,14]-conantokin G treatment group was 534.3±101.8, P=0.093; time spent in white chamber in 60 pmol [Glu3,4,7,10,14]-conantokin G treatment group was 426.1±123.9 , P=0.016 ; time spent in white chamber in 120 pmol [Glu3,4,7,10,14]-conantokin G treatment group was 378.1±250.9,P=0.046). Total distance moved, shuttle times, total distance moved in center area, total time spent in center area, distance moved in white chamber and time spent in white chamber showed no significant difference among each group (P>0.05).3.3 Correlation analysis among each indexPartial correlation analysis showed that: time spent in the white chamber and time spent in the center area of white chamber showed correlation relationship(r=0.452, P<0.05); total distance moved and shuttle times(r=0.439, P<0.05) and total distance moved in the center area (r=0.574, P<0.05) and distance moved in the center area of white chamber (r=0.501, P<0.05) showed correlation relationship. Among the five indexes reflect the exploring activity, shuttle times and total distance moved in the center area showed correlation relationship (r=0.505, P <0.05) , shuttle times and total time spent in the center area showed correlation relationship (r=0.274, P<0.05), shuttle times and distance moved in the center area of white chamber showed correlation relationship (r=0.454, P<0.05) , shuttle times and time spent in the center area of white chamber showed no correlation relationship (r=0.191, P=0.132) ; total distance moved in the center area and total time spent in the center area showed correlation relationship (r=0.718, P<0.05) , total distance moved in the center area and distance moved in the center area of white chamber showed correlation relationship (r=0.827, P<0.05), total distance moved in the center area and time spent in the center area of white chamber showed correlation relationship (r=0.403, P<0.05); total time spent in the center area and distance moved in the center area of white chamber showed correlation relationship(r=0.679, P<0.05), total time spent in the center area and time spent in the center area of white chamber showed correlation relationship (r=0.801, P<0.05), distance moved in the center area of white chamber and time spent in the center area of white chamber showed correlation relationship (r=0.641,P<0.05).3.4 Change of the p-ERK1/2 in striatum, PFC and hippocampus of mice after morphine CPP expression attenuated by Glu-ConGThe expression of ERK1/2 among each groups showed no significant difference (P>0.05), p-ERK1/2 increased in morphine group in hippocampus , prefrontal cortex and striatum brain regions (P<0.05) ; compared with morphine group, in hippocampus and straitum, the expression of p-ERK1/2 in 30, 60 and 120pmol Glu-ConG treatment groups and saline group decreased (P<0.05), in prefrontal cortex, the expression of p-ERK1/2 in 60 and 120pmol Glu-ConG treatment groups and saline group decreased (P<0.05) .4. Conclusions1. The mice preferred black chamber naturally, 5mg/kg morphine(i.p.)could induced a significant CPP to drug-paired side;2. Glu-Con G(i.c.v.) inhibited the morphine induced CPP in a dose-related manner, the effective dose was 30~60pmol(equals 0.0015~0.003mg/kg), but did not effected the locomotor activity and exploring activity;3. Morphine increased the p-ERK1/2 expression in hippocampus, prefrontal cortex and striatum, but the ERK1/2 expression do not change.4. Glu-ConG blocked the expression of p-ERK1/2 in hippocampus, prefrontal cortex and striatum in a dose-related manner, which supplies more evidences to explain the NMDA receptors and p-ERK1/2 are involved in the morphine dependence.

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